The cell is the most fundamental unit of living organisms, whether animal or plant. The study of its structure and composition, and how its various constituents function, lends valuable insight into the complex processes that occur in integrated biological systems. This requires techniques that allow investigation of cell samples to be conducted in real-time, non-invasively, and in solutions that mimic physiological conditions so that cell functionality is retained.
Optical microscopy (using visible light) has been widely applied to study live cells. However, the resolution is limited by diffraction to about 200–250 nm. For more detailed study, one commonly used method is electron microscopy, where it is possible to obtain images with 10 nm resolution, but the sample needs to be fixed prior to imaging. Hence, it is not possible to use an electron microscope to study living cells.
Another possible high resolution technique is based on the use of scanning probe microscopy (SPM), in which a sharp probe tip is scanned in close proximity to the sample understudy. The consequent interactions and thus the chemical/physical properties of the sample can be plotted as a function of the tip's position with respect to the sample, to generate a profile of this measured interaction. Members of the SPM family that are commonly applied to biological imaging are atomic free microscopy (AFM), scanning ion-conductance microscopy (SICM) and scanning near-field optical microscopy (SNOM).
In SNOM, light is normally coupled down a fibre-optic probe with an output aperture of sub-wavelength dimensions, which is scanned above the sample surface. Interaction forces between the tip and sample are used to maintain their separation at less than the sub-wavelength dimensions of the aperture. This arrangement allows simultaneous generation of optical and topographic images whose resolution depends on the size of the output aperture and the size of the tip respectively. As in far field optical microscopy, all contrast mechanisms are available in SNOM, and in particular chemical imaging is possible by the use of fluorescent labels. However, while it is straightforward to fabricate probes with smaller apertures, achieving smaller tip-sample separations in liquid (<60 nm) is difficult because of the problems in obtaining a reliable method of controlling the probe-sample distance. This is due to damping of the oscillations of the probe used in the feedback mechanism.
In SICM, an electrolyte-filled, glass micropipette is scanned over the surface of a sample bathed in an electrolytic solution; see Hansma et al (1989) Science 243:641–3. The pipette-sample separation is maintained at a constant value by controlling the ion-current that flows via the pipette aperture. The flow is between two electrodes: one inside the pipette and another outside in the electrolyte solution. For an applied bias between the electrodes, the ion-current signal depends on a combination of the micropipette's resistance (RP) and the access resistance (RAC) which is the resistance along the convergent paths from the bath to the micropipette opening. RP depends on the tip diameter and cone angle of the micropipette, whereas RAC displays complicated dependence on the sample's electrochemical properties, geometry and separation from the probe. It is RAC that lends ion-current sensitivity to the pipette-sample separation and allows its exploitation in maintaining the distance such that contact does not occur.
The optimum tip-sample separation that has allowed SICM to be established as a non-contact profiling method for elaborated surfaces, is equal to one-half of the tip diameter; see Korchev et al (1997), J. Microsc. 188:17–23, and also Biophys. J. 73:653–8. The tip's output is used to generate topographic features and/or images of the local ion-currents flowing through pores on the sample surface. The spatial resolution achievable using SICM is dependent on the size of the tip aperture, and is typically between 50 nm and 1.5 μm. This produces a corresponding resolution.